Solid-supported organic synthesis has recently entered a renaissance due to the emergence of the field of combinatorial chemistry, aimed at rapidly synthesizing vast libraries of compounds for biological testing (Moos, W. H.; et al., Annual Reports in Medicinal Chemistry (Bristol, J. A. editor), 28:315 (1993)). Most of these libraries are produced through solid-supported organic synthesis and consist of peptides or peptide-like compounds (Zuckermann, R. N.; et al., J. Am. Chem. Soc., 114:10646 (1992); Houghten, R. A.; et al., Nature, 354:84 (1991)). Some groups have begun to produce chemical libraries that do not rely on the peptide backbone (Lebl, M.; et al., Int. J. Pept. Protein Res., 41:201 (1993); Dewitt, S. H.; et al., Proc. Natl. Acad. Sci. USA, 90:6909 (1993)). The major advantage in producing chemical libraries by solid-supported synthesis over traditional solution synthesis is the relative ease in separating starting material from product. This rapid ability to separate staring material from product can also make the library's production through automation that much easier.
However, in the field of solid-supported peptide synthesis, the methods of separation have been filtration and centrifugation. The physical separation of the support from the solubilized components of the reaction mixture has primarily been accomplished by filtration through a glass or polymer filter (Wolfe, H. R.; Wilk R. R., Peptide Research, 2:352 (1989); Knapp, D. R.; et al., Int. J. Peptide Protein Res., 42:259 (1993)). Centrifugation has also been used as a means of separation (Anderson, N. G.; et al., J. In Innovation and Perspectives in Solid Phase Synthesis, Oxford, In Press (1993); Lebl. M., et al., U.S. Pat. No. 5,202,418). Although filtration has been the method of choice in both solid-phase peptide and nucleotide synthesis, it does have limitations that warrant the development of new approaches. One such limitation is the difficulty of automating the simultaneous washing and filtration of hundreds of small scale solid-phase reactions. Another problem is the tendency for a percentage of the filters to get clogged over time with precipitated reagent which makes the use of filtration in automated procedures less reliable.
Automation of bioassays and organic syntheses is desirable to improve quality control in order to insure uniformity of results. A field that has had some success in automating some of its techniques is immunodiagnostics. Antibody-bound paramagnetic beads can be exposed to a magnetic field to separate antibody-bound antigen from unbound antigen in immunoassays (Okada, M., et al., U.S. Pat. No. 5,320,944; Ugelstad, J., et al., WO 83/03920; Fjeld, J. G., et al., J. Immunol. Methods, 109:1 (1988)), each of which is incorporated herein by reference. Assays directly measuring the radiolabeled ligand-protein complex after it has been separate from unbound material are termed radioimmunoassays. Alternatively, in a method termed scintillation proximity assay, the extent of binding is determined by measuring the intensity of fluorescence released when the radiolabel affects a fluorescent molecule bound to another particle (H. Hat; U.S. Pat. No. 4,271,139) incorporated herein by reference. However, the loading capacity of the support systems currently used is limited. Further, non-specific binding of unbound reactants to the support can yield incorrectly elevated values.
Magnetic separation methods have also been applied successfully in cell sorting (Treleaven, J. G.; Lancet, 14:70 (1984); Miltenyi, S.; et al.; Cytometry, 11:231 (1990); Padmanabhan, R., et al., Analytical Biochem., 170:341 (1988)). A definite advantage that magnetic separation has over simple filtration is the ability to separate out particles in small reaction volumes. As with immunoassays, the loading capacity of the support systems currently used is limited.
Unlike immunodiagnostic and cell sorting technologies, the use of magnetic separation in the field of solid-supported organic chemistry has been slow in coming due to the instability in organic solvents such as dimethylformamide and methylene chloride exhibited by the currently available supports. Upon exposure to these solvents, typical polymer coated magnetic beads dissolve. Silica coated magnetic beads are more stable to these solvents but lack the loading capacity (typically&lt;0.2 mmoles/gram) and acid stability that make them practical for organic synthesis (Benner S. A., U.S. Pat. No. 4,638,032).
One way to increase paramagnetic polymer beds stability in organic solvent is to enhance the levels of cross-linking of the polymer around the magnetite core to make macroporous polymer particles (Ugelstad, J., et al., U.S. Pat. No. 4,774,265; Wang, C. H. J., and Shah, D. O., U.S. Pat. No. 5,283,079), each of which is incorporated herein by reference. A major problem with this approach is that higher levels of cross-linking reduce the extent to which the support expands and contracts and also affects the level of reactivity. Work by Regen using electron spin resonance (ESR) spectroscopy of nitroxide radical probes showed that the bound substrate was more restricted than a substrate dissolved in the swollen particle (Regen, S. L. J. Am. Chem. Soc., 96:5275 (1974); Regen, S. L., Macromolecules, 8: 689 (1975)). In addition, Regen provided evidence that with greater cross-linking, which translates to less expansion of the support, the internal viscosity of the solvent increased.
Again looking to the non-magnetic solid-phase support field, one can dramatically enhance the mobility of the bound substrate even with high levels of cross-linking of the support by attaching a long polyethylene glycol (MW=2000-3000 daltons) spacer arm which acts as a linker between the bound molecule and the support (Bayer, E., and Rapp, W., U.S. Pat. No. 4,908,405; Barany, G., et al., WO 92/04384), each of which is incorporated herein by reference. However, previous methods required multiple, complex reactions to achieve coupling of the spacer arm to the support.
Clearly, what is needed is an organic solvent stable support which can achieve high loading capacities, which is easily manufactured, and which is useful in automated assays and solid phase organic syntheses.